Liquid chromatography is widely used to prepare and analyze biological samples, and to purify components of biological samples from contaminants. In liquid chromatography, a sample is suspended in a mobile phase, such as a buffer, which is passed over a stationary phase, such as a column. The binding of the sample, or particular components thereof, to the stationary phase, and the subsequent elution of the sample back into the mobile phase is controlled by altering the chemical environment to which the sample is exposed. This is typically done by changing the makeup of the mobile phase. Two or more buffers, containing disparate solute concentrations or having different pHs, for example, can be mixed together in gradually varying portions, and the resulting mixture can be applied to the column to cause binding and elution of the sample at the desired times. The parts of a liquid chromatography system that draw multiple buffers (or other liquids) from their source containers, mix the buffers together, and deliver the mixture to the column are together an example of a fluid delivery system.
In order to attain the desired level of control of sample binding and elution in liquid chromatography, the fluid delivery system must be properly calibrated. For example, the actual portion of each buffer in the mixture should be compared with the desired portion, across the full range of portions that might be encountered, and any disparities should be reduced or eliminated. If mixing can be turned off and the buffers can be kept separate while passing through the system, then the amounts of buffer that arrive at the column (or the rates at which they arrive) can be measured. However, this is not feasible in many fluid delivery systems, where buffer intake and mixing are tightly coupled. Alternatively, the rates at which the source containers decrease in mass can be measured, but this requires balances and other equipment that is typically not available in liquid chromatography setups.
The concentrations of biological species, such as proteins and nucleic acids, that pass through the column are often monitored using optical absorbance. Ultraviolet, visible, or infrared light is made incident upon the mobile phase as it exits the column, and the absorbance of the mobile phase is monitored at one or more wavelengths in real time. If the extinction coefficient of a species at a particular wavelength is known, then its concentration can be inferred. Absorbance measurements are not limited to biological species, can reflect components of the buffers that are mixed together in the fluid delivery system, and can be made upstream of (or in the absence of) the column.